A comparative study of PCNA and Ki-67 expression in dental follicle, dentigerous cyst, unicystic ameloblastoma and ameloblastoma

Various cell proliferation markers are used as diagnostic and prognostic tools in oral lesions. Simultaneous evaluation of these markers can increase the precision of estimation of the proliferative status of different tissues. In this study we investigated the expression of PCNA and Ki-67 as markers of cell proliferation in 15 paraffin embedded samples of each dental follicle, dentigerous cyst, unicystic ameloblastoma and ameloblastoma belonging to a total of 30 male and 30 female paients using immunohistochemistry method. Expression levels based on the intensity and the percentage of stained cells was separately analyzed for each marker with chi-square test, the results of which were significant for the two markers (P<0.05). The correlation coefficient between the two markers was found to be 0.88. A significant difference in the expression of Ki-67 and PCNA was observed in the four types of studied lesions.

ental follicle originates from odontogenic ectomesenchyme and is one of the components of the tooth germ. It is composed of two parts including the crown and the root which itself includes enamel organ, dental papilla, and dental follicle. The developing tooth germ is surrounded by a dense embryonic connective tissue known as dental sac or dental follicle (1,2). Dentigerous cyst as the most common developmental odontogenic cyst, accounts for 20% of all epithelium-lined jaw cysts. The cyst encloses an undeveloped crown and attaches to the tooth at cemento-enamel junction (CEJ). Although the pathogenesis of the cyst is unknown, it obviously develops due to fluid accumulation between the reduced enamel epithelium and the tooth crown. The cyst is often found at the age of 10 to 30 years and is more common in males with male to female ratio of 1.  (1,2). Ameloblastoma is an odontogenic tumor, which accounts for 1% of head and neck tumors. It grows slowly without any pain and symptom, and despite the benign nature of the lesion, localized invasion has been reported. The lesion tends to have high recurrence and metastases in rare cases.
Unicystic Ameloblastoma (UA) was first described in 1977 by Robinson and Martinez (3). UA is most commonly observed in the mandibular molar-ramus region. The lesion is in some cases associated with impacted third molars. It is most commonly encountered at the second decade of life.
There are different methods for evaluating oral lesions in which certain proteins and other substances in the lesion are directly and indirectly measured (4,5). The 395-kD protein -Ki-67 -is expressed in proliferating cells and during DNA synthesis and immediately disappears after mitosis (6,7). This antigen is preferentially expressed during late G1, S, G2 and M phase, whereas resting, noncycling cells (G0 phase) lack Ki-67 expression.
Because of its absence in quiescent cells (G0 phase), this protein developed into a widely used tumor marker in the fields of research and pathology (8).
Ki-67 is of prognostic value for many types of malignant tumors (9). Proliferating nuclear cell antigen (PCNA) is known as an important protein in DNA synthesis and repair (6,7). This nuclear non-histone protein is an accessory protein for DNA polymerase alpha, an essential factor for DNA replication and repair. This protein is elevated during the G1/S phase (10).  (11)(12)(13). It is known that Ki-67 is a more specific marker for the proliferation of ameloblastic tumor cells (4).
Therefore, it seems that simultaneous evaluation of these two markers can be a precise estimation for the proliferative function of tissues and different tumors that can also be helpful in determining progression, aggressiveness and prognosis of the lesions. Hence, the present study aimed to investigate the expression of Ki-67 and PCNA in dental follicle, dentigerous cyst, unicystic ameloblastoma and ameloblastoma using immunohistochemistry method.

Materials and Methods
The present analytical cross-sectional study The sum of these two was then considered as the final score (9); data were analyzed by SPSS18 statistical software using Mann-Whitney, Pearson correlation, Chi-Square and One Way ANOVA tests. Quantitative data are presented as mean (± Standard Deviation) and qualitative data as frequency.

Results
Out of 60 samples studied, 30 patients were male and 30 were female. The mean age of patients was 19.57 (± 11.35) years, ranging from 5 to 59 years. The expression of Ki-67 (Fig 1) and PCNA (Fig 2) in dental follicle, dentigerous cyst, unicystic ameloblastoma and ameloblastoma based on the staining intensity and the percentage of stained cells are presented in Tables 1 and 2 respectively.

Discussion
In the present study, dental follicle, dentigerous

Aknowlegement:
This study was a student thesis in Babol dental school and was financially supported by vice chancellery for research of Babol university of medical sciences. The authors wish to thank Mr Mohsen Aghajanpour for staining the samples.